Anti-Infective and Anti-Cancer Properties of the Annona Species: Their Ethnomedicinal Uses, Alkaloid Diversity, and Pharmacological Activities

Annona species have been a valuable source of anti-infective and anticancer agents. However, only limited evaluations of their alkaloids have been carried out. This review collates and evaluates the biological data from extracts and purified isolates for their anti-infective and anti-cancer activities. An isoquinoline backbone is a major structural alkaloid moiety of the Annona genus, and more than 83 alkaloids have been isolated from this genus alone. Crude extracts of Annona genus are reported with moderate activities against Plasmodium falciparum showing larvicidal activities. However, no pure compounds from the Annona genus were tested against the parasite. The methanol extract of Annona muricata showed apparent antimicrobial activities. The isolated alkaloids from this genus including liriodenine, anonaine, asimilobine showed sensitivity against Staphylococcus epidermidis. Other alkaloids such as (+)-Xylopine and isocoreximine indicated significant anti-cancer activity against A549 and K-562 cell lines, respectively. This review revealed that the alkaloids from Annona genus are rich in structural diversity and pharmacological activities. Further exploration of this genus and their alkaloids has potential for developing novel anti-infective and anticancer drugs.


Introduction
Annona is one of the 129 genera of the Annonaceae family and contains 119 species with eight species grown for commercial uses [1,2]. Most of the species grow in tropical regions; e.g., the soursop fruit tree (Annona muricata) is cultivated commercially and is widespread in the West Indies, North and South Americas, Africa, the Pacific Islands, and Southeast Asia. Annona species have been used as medicines by indigenous people for a wide range of disorders including parasitic infections, inflammation, diabetes, and cancer [3]. The phytochemical investigation of this plant genus has revealed the presence of acetogenins, alkaloids, essential oils, flavonoids, terpenoids, and other chemical classes [4,5]. Acetogenins (ACGs) are the major constituents of the Annona genre and examples were found to possess a variety of pharmacological properties including as antitumor, immunosuppressive, pesticidal, antiprotozoal, antimicrobial, antimalarial, anthelmintic, and antiviral agents, with some being commercially developed for the treatment of oral herpes and treating infestations of head lice, fleas, and ticks [5,6]. However, the available phytochemistry, including

Phytochemical Studies of Secondary Metabolites of Annona Genus
The juicy pulp of the fruit is often a good source of sugar, vitamins, minerals, and phenolic intake. For example, the dried pulp of Annona muricata contains 68% sugars for every 100 g containing 1.0 g protein, 0.97 g fat, 1.28 niacin, and 29 mg ascorbic acid. Moreover, it could supply 3 g of phenolic substances for every 100 g of pulp [9,17]. The 20th century reported preliminary examinations of the Annona plants of the leaves, fruits, and seeds. Since the 1980s, with the advent of pursuing anti-cancer drug leads from medicinal plants, acetogenin was isolated from the Annona genus based on its promising anti-cancer activity. For example, a recent acetogenin, squamocin P, isolated from A. squamosa, possessed significant anticancer activity against SMMC 7721/T, MCF-7/ADR, A549/T with IC50 values of 0.435, 3.34, 6.32 μM, respectively, with the positive control cisplatin having higher IC50 values of 198.85, 178.87, and 219.33 μM against SMMC 7721/T, MCF-7/ADR, and A549/T, respectively. While this encouraged investigations into this species, they were confined to this one polyketide compound, at the expense of other components present. Figure  1a shows the number of compounds isolated from each plant part of Annona muricata. In the previous phytochemical studies of Annona muricata, around 127 compounds were isolated, in which almost 90% were acetogenins (Figure 1b) [18]. Acetogenins from the Annona genus were reviewed together with other genus in the same family Annonaceae [8,[19][20][21][22][23], which covered the isolation, molecular properties, and biosynthesis of their pharmacological activities. Here, we collected records on alkaloids which were isolated in the Annona plant genus from 1960-2019 (Table 1). The alkaloids present have been of interest since the first, annonaine (8, Figure 2), was isolated in 1931 from the stem bark of Annona muricata L collected in the Philippines [24]. Table 1 shows the alkaloids isolated from the specific plants of each species and their structures are presented in Figure 2.   [29,30]  Acetogenins from the Annona genus were reviewed together with other genus in the same family Annonaceae [8,[19][20][21][22][23], which covered the isolation, molecular properties, and biosynthesis of their pharmacological activities. Here, we collected records on alkaloids which were isolated in the Annona plant genus from 1960-2019 (Table 1). The alkaloids present have been of interest since the first, annonaine (8, Figure 2), was isolated in 1931 from the stem bark of Annona muricata L. collected in the Philippines [24]. Table 1 shows the alkaloids isolated from the specific plants of each species and their structures are presented in Figure 2.

Anti-Infective Alkaloids from the Genus Annona
Plants from the genus Annona plants have been used in traditional medication for the treatment of both infectious and non-infectious diseases. This led to the pharmacological and chemical screening of numerous species to confirm these pharmacological claims and to isolate the compounds which might be responsible for these activities. The Annona genus has been studied for activity against parasites, cancer, and as anti-oxidant agents.

Antiprotozoal Activities
Ethnopharmacological studies have revealed the Annona species Annona crassiflora, A. muricata, A. senegalensis, and A. squamosa were prescribed in malarial fever therapy. Further studies revealed leaf extract from A. crassiflora was rich in flavonoids and alkaloids, and was able to reduce the Plamsodium berghei NK65 infection level in mice by 57-75% with a daily dosage of 12.5 μg/kg/day [62]. Another study of the crude methanol extract of A. squamosa indicated moderate activity against Plasmodium falciparum 3D7 with an IC50 value of 30 μg/mL compared to the chloroquine control, which gave an IC50 value of 0.021 μg/mL [63]. Moderate anti-plasmodium activity was also shown using crude extracts of A. muricata (Table 2).
In an animal model test, an aqueous leaf extract of A. muricata showed a dose dependent antimalarial effect with the highest inhibition of 85.61% observed from a 1000 μg/kg dose. However, the treatment was unable to completely cure the mice, but prolonged the survival time [64]. An essential oil extract of A. squamosa demonstrated inhibition against the erythrocitic stages of P. falciparum, against epimastigotes forms of T. cruzi and against trypomastigotes forms of T. cruzi with IC50 values of 14.7, 16.2, and 12.7 μg/mL, respectively [65].
Although there was a limited record regarding traditional uses of Annona plants to treat other parasitic protozoal infections, e.g., leishmaniasis and trypanosomiasis, several crude extracts of Annona plant (A. muricata) were also tested against L. amazonensis, L. braziliensis, L. Donovani, and T. cruzi ( Table 2). The crude ethyl acetate extract from the leaves of A. muricata indicated potent activity against L. amazonensis, L. braziliensis, L. Donovani, and T. cruzi with IC50 values of 10-25 μg/mL. A different strategy to control malarial infection involves controlling its vector. Past larvicidal studies have indicated that the crude methanol extract from the bark of A. squamosa resulted in 100% mortality of Anopheles subpictus (which carry human malaria parasites) at 500 μg/mL [66]. The extract from the stem and root bark were even more toxic toward malarial larvae (Anopheles gambiaes.s. Giles) with 50% mortality at 24 and 21 μg/mL, respectively [67].    Annona diversifolia

Plant Parts Location Isolated Alkaloids
Annona squamosa

Anti-Infective Alkaloids from the Genus Annona
Plants from the genus Annona plants have been used in traditional medication for the treatment of both infectious and non-infectious diseases. This led to the pharmacological and chemical screening of numerous species to confirm these pharmacological claims and to isolate the compounds which might be responsible for these activities. The Annona genus has been studied for activity against parasites, cancer, and as anti-oxidant agents.

Antiprotozoal Activities
Ethnopharmacological studies have revealed the Annona species Annona crassiflora, A. muricata, A. senegalensis, and A. squamosa were prescribed in malarial fever therapy. Further studies revealed leaf extract from A. crassiflora was rich in flavonoids and alkaloids, and was able to reduce the Plamsodium berghei NK65 infection level in mice by 57-75% with a daily dosage of 12.5 µg/kg/day [62]. Another study of the crude methanol extract of A. squamosa indicated moderate activity against Plasmodium falciparum 3D7 with an IC 50 value of 30 µg/mL compared to the chloroquine control, which gave an IC 50 value of 0.021 µg/mL [63]. Moderate anti-plasmodium activity was also shown using crude extracts of A. muricata (Table 2).
In an animal model test, an aqueous leaf extract of A. muricata showed a dose dependent antimalarial effect with the highest inhibition of 85.61% observed from a 1000 µg/kg dose. However, the treatment was unable to completely cure the mice, but prolonged the survival time [64]. An essential oil extract of A. squamosa demonstrated inhibition against the erythrocitic stages of P. falciparum, against epimastigotes forms of T. cruzi and against trypomastigotes forms of T. cruzi with IC 50 values of 14.7, 16.2, and 12.7 µg/mL, respectively [65].
Although there was a limited record regarding traditional uses of Annona plants to treat other parasitic protozoal infections, e.g., leishmaniasis and trypanosomiasis, several crude extracts of Annona plant (A. muricata) were also tested against L. amazonensis, L. braziliensis, L. Donovani, and T. cruzi ( Table 2). The crude ethyl acetate extract from the leaves of A. muricata indicated potent activity against L. amazonensis, L. braziliensis, L. Donovani, and T. cruzi with IC 50 values of 10-25 µg/mL. A different strategy to control malarial infection involves controlling its vector. Past larvicidal studies have indicated that the crude methanol extract from the bark of A. squamosa resulted in 100% mortality of Anopheles subpictus (which carry human malaria parasites) at 500 µg/mL [66]. The extract from the stem and root bark were even more toxic toward malarial larvae (Anopheles gambiaes.s. Giles) with 50% mortality at 24 and 21 µg/mL, respectively [67]. The same protocol was applied to other disease vectors, including Aedes (dengue virus vector) and Culex (encephalitis virus). For example, the seeds of Annonas pecies were generally reported to be toxic with LC 50 values <1 µg/mL against both Aedes and Culex larvae (Table 3). These results demonstrated that the Annona plants can be used for controlling the vector especially in rural areas where modern, and likely more expensive, vector controls were limited.
Despite numerous alkaloids being isolated from Annona species, reports detailing pharmacological studies on single compounds remains limited. There are reports on the same alkaloids being isolated from different plant genus. For example, (+)-reticuline 19 was isolated from Croton linearis and was previously shown to possess a weak antriprotozoal activity against Lesihmania infatum with IC 50 values of 148.0 ± 1.2 µM [70]. Asimilobine 9 and isoboldine 17 isolated from the bark of Beilschmiedia alloiophylla (Costa Rica) possessed anti-leishmanial activity with IC 50 values of 29.8 ± 1.5 µM and 50.0 ± 4.0 µM, respectively [71]. A previous study on the leaves and fruits of Annona mucosa (Brazil) produced liriodenine 11, which was highly active against Leishmania amazonensis with an IC 50 value of 1.43 ± 0.58 µg/mL and was moderately active against Leishmania braziliensis with an IC 50 value of 55.92 ± 3.55 µg/mL [72].

Antimicrobial Activities
Traditionally, Annona plants have been prepared for use against infection related diseases, such as ulcer, dysentery, and boils, and therefore became a driving force for conducting anti-microbial studies against common bacteria; preliminary results on the crude extracts are shown in Table 4. In general, the crude extract possessed moderate to inactive anti-microbial values ranging from 6.25-4096 µg/mL. Most of the studies were based on the anti-microbial activity of crude extracts with no separate non-polar to polar fractions tested or individual constituents isolated. Therefore, further investigations are required to substantiate the traditional claims for these Annona plants by the isolation and identification of individual constituents. As a result, discussion here is confined to the anti-microbial activities from isolated alkaloid constituents (Table 5). A. muricata, A. squamosa, A. cherimola, and A. ambotay showed reasonable antimicrobial activities, whereas A. reticulata did not present antimicrobial activity, with reported MIC values of more than 1000 µg/mL against Bacillus cereus, Staphylococcus aureus [77]. Antimicrobial testing of the methanol extract of A. squamosa fruit against multidrug resistant MRSA reported MIC values of 5000 µg/mL, but no information was given against ESBLEC (extended-spectrum beta-lactamase producing E. coli), CRPA (carbapenem-resistant P. aeruginosa) and MDRAB (multidrug-resistant A. baumannii) [78]. The benzoquinoline alkaloid, anonaine 8, indicated comparable anti-microbial activities with positive control, with the exception against Staphylococcus aureus. Another study reported annoquinone A, isolated from A. Montana, possessed anti-microbial activity against Bacillus subtilis and Micrococcus luteus with IC 50 value of 10, 10 µg/mL, respectively [79].   Previous studies using alkaloid samples from sources other than Annona revealed, (−)-asimilobine 9 isolated from the bark of Beilschmiedia alloiophylla (Costa Rica) and B. kunstleri (Malaysia) indicated anti-fungal activity with an IC 50 value of 16.0 µg/mL [71]. (−)-Stepholidine 20 isolated from rattan stem of Fibraurea recisa had antifungal activity against drug resistant Candida albicans SM372, Candida krusei KM066, Candida parapsilosis SM304160, Cryptococcus neofarms SM9406204 with similar MIC value of 320 µg/mL [91]. Alkaloid (−)-roemerine 49 from the same stem indicated significant inhibition of C. albican transition from yeast to hyphae in a dose dependent manner [92]. Glaucine 68 isolated from the aerial component of Glaucium oxylobum showed moderate skin anti-fungal activities against Microsporum canis, Microsporum gypseum, and Trichophyton mentagrophytes [93]. Antifungal activities of the non-Annona isolated alkaloids were evaluated against non-pathogenic fungi including liriodenine 11 from the wood of Michelia formosa which indicated a low activity against several wood decaying fungi both white and brown rot-fungi, Lenzites betulina, Trametes versicolor, Laetiporus sulphureus, Gloeophyllum trabeum, and Fomitopsis pinicola [94]. Similar alkaloids were also previously evaluated against pathogenic bacteria, including liriodenine from the roots of Zanthoxylum nitidum which showed a good antimicrobial activity against MRSA with MIC value of 93.8 µg/mL [95]. Liriodenine 11 from the stem of Mitrephira glabra Scheff was active against non-pathogenic bacteria, Micrococcus luteus, Mycobacterium sinegmatis, Saccharomyces cerevisae, and Aspergilus niger with an MIC value of 6.3, 12, 12, and 25 µg/mL, respectively [96].

Anticancer Alkaloids Present in the Genus Annona
In addition to the above antiprotozoal and antimicrobial activities, both the crude extracts from annona plants and the individual alkaloids have shown potent anticancer/antitumour activities.Many crude extracts of Annona species showed significant anti-cancer activities, but most of the bioactive constituents present in those crude extracts were acetogenins, fatty acids, and peptides [7]. However, wherever studied, it was known that some aporphine alkaloids, especially (−)-roemerine 49, which was isolated from the leaves of the wild custard apple, improved the response produced by vinblastine against multidrug-resistant KB-V1 or KB-3 cells (ED 50 > 20 µg/mL). This alkaloid appears to function by interacting with P-glycoprotein in the multidrug-resistant KB-V1 cell membrane vesicles [59]. The leaves of Annona muricata also showed potency to reduce gastric lesion, to expel parasitic worms and, moreover, the crude extract from the bark possessed anti-viral activity against herpes simplex virus type 1. The extracts and compounds also showed anticancer activities against breast cancer. Alkaloids,  [34].
Although most of the alkaloids isolated from Annona species were reported with no anticancer activity data, there were cytotoxicity activity data on similar molecules obtained from non-Annona genus (Table 6). Interestingly, annomontine 54, a carbolated pyrimidine alkaloid was previously reported from the marine sponge Acanthostrongylophora ingens collected from Indonesian water. The alkaloid possessed pronounced anticancer activity against mouse lymphoma L5178Y compared to a standard control kahallide F [97]. The oxoaporphine alkaloid, liriodenine 11, was found in at least in twenty different species, ranging across flowering plants but mostly in annonaceae family. The alkaloid isolated from Brazilian Guatteria blepharophylla stem bark possessed anticancer activity against MCF-7 cell line with a more potent result compared to a standard drug doxorubicin with TGI value of 36.67 compared to 46.04 µM [98].

Conclusions
This review presents the ethnomedicinal, alkaloidal and biological, properties of Annona species with respect to reported anti-infective and anti-cancer activities. The Anonna species: A. muricata (soursop), A. squamosa (custard apple), A. senegalensis (wild custard apple), and A. cherimola (cherimola) are renowned traditionally for their anti-tumor properties. Among these, A. muricata is widely studied and has shown broad range of biological activities including anti-protozoal, anti-cancer, anti-tumour, antimicrobial, and antiparasitic properties. This species has also produced several patents and commercial products. Investigations into extracts from the leaves, bark, fruit, and seeds of this plant genus have found terpenoids, steroids, flavonoids, cardiac glycosides, tannins, phenols, sugars, fatty acids, acetogenins, and alkaloids. As many as 200 phytochemicals belonging to these chemotypes have been identified and isolated from this Annona muricata species alone, with the most important being acetogenins, phenols and anonaine alkaloids. Anonaine and its structurally related alkaloids were the most abundant and commonly available alkaloids in Annonaceae family. The oxoaporphine alkaloid, liriodenine, was found in at least in twenty different species, ranging across flowering plants but mostly in annonaceae family. The alkaloids from Annona species have rarely been explored for their medicinal applications. However, wherever studied, Annona alkaloids have been reported to possess anti-inflammatory, anti-cancer, antitumor, anti-HIV, antiprotozoal, antiparasitic, antidiabetic, analgesics, gastroprotective, antihypertensive, hepatoprotective, nephroprotective, and neuroprotective properties. Amongst these broad-ranging properties, anti-cancer and anti-tumour activities of both the crude extracts and alkaloids is commendable. Most interesting and noteworthy of this Annona genera is that the pharmacological properties accentuate the ethnomedicinal utilization of this plant, as well as its usefulness in the agrifood sector. Liriodenine, annonaine, glaucine and cleistopholine showed potent anti-cancer, anti-tumour, and cytotoxicity activities against many human cancer cell lines, and it is worthwhile to pursue detailed clinical investigations of these alkaloids. To the best of our knowledge, there is no clinical study that was successfully completed on the extracts rich in acetogenins or alkaloids. In this respect, it is also necessary to conduct scientific studies to establish optimal and safe doses of consumption of both the plant extracts and their phytochemicals especially alkaloids. This is because the use of the Annona plants is popular not only in Indonesia, but wide across the tropical countries.